This invention relates to the preparation by thin-layer chromatographic fractionation of an aqueous extract from seeds of the plant Paullinia cupana H.B.K. (guarana) which possesses biological activity, more particularly an ability to inhibit platelet aggregation in mammalian blood and to deaggregate platelet aggregates induced by adenosine diphosphate (ADP) and/or arachidonic acid. Formation of thromboxane B.sub.2 is also inhibited by the extract of this invention, thus indicating utility in the treatment of thrombosis and other vascular disorders involving platelet aggregation.
Blood platelets are small cells present in large numbers in mammalian blood and are vital in arresting bleeding by aggregating to form a platelet plug. When bleeding does not occur, internal injury to a blood vessel wall may cause formation of a platelet plug which is called a thrombus. Thrombus formation bay block the flow of blood to an organ and cause infarction, a condition termed thrombosis. Thrombosis is considered to be a contributing cause to strokes, pulmonary embolisms, and myocardial infarctions. It is thus believed that ability to inhibit platelet aggregation and to deaggregate platelet aggregates would be of great benefit in reducing the adverse effects of thrombotic episodes.
The tropical plant guarana has been harvested in the Amazon basin for many years. Treatment of the seeds of this plant, and uses thereof in the form of syrups, extracts and distillates as flavoring agents and as a source of caffeine in the soft-drink industry are described by A. R. Henman in Guarana (Paullinia Cupana Var. Sorbilis): Ecological And Social Perspectives On An Economic Plant Of The Central Amazon Basin, Jour. of Ethnopharmacology 6 (1982) 311-338. It is stated therein that recently ". . . claims have been made for guarana's suitability as a tea and coffee substitute, particularly for those suffering from cardiovascular afflictions, and this could be related to the counterbalance given to the stimulant alkaloids in the drug by the saponins which are so well represented in the plant family which includes guarana (the Sapindaceae)."
F. Belliardo et al., in HPLC Determination of Caffeine and Theophylline in Paullinia cupana Kunth (Guarana) and Cola spp. Samples, Z. Lebens Unters Forsch (1985) 180:398-401 (Springer-Verlag), report quantitative estimations of caffeine and theophylline in guarana, commercial guarana and cola samples, by a reversed-phase, high-performance liquid-chromatographic method. Five different extraction procedures are disclosed, four of which involved, in general, suspending a sample in distilled water containing ammonia, hydrochloric acid, or ammonia plus chloroform; or in ethanol (70%), followed by boiling or heating under reflux, extraction with chloroform, drying the chloroform extracts over magnesium sulfate, evaporating to dryness in vacuo, dissolving the residue in a 1:1 methanol-water mixture acidified with hydrochloric acid, and diluting to volume. The fifth method, which gave the best recovery, comprised suspending a sample in distilled water (20 ml) containing 1 drop of concentrated HCl and 10 ml. of an aqueous solution of 8-chlorotheophylline (0.376 mg/ml) as an internal standard, heating under reflux for 15 minutes, cooling, centrifuging (4000 rpm for 10 minutes), separating the aqueous phase, washing the residue with aqueous acid solution, and diluting the combined aqueous solutions with H.sub.2 O to a volume of 100 ml. Before HPLC analysis the solution was filtered. Analytical determination was done with a Perkin-Elmer Series 3B liquid chromatograph.
Synthetic compounds stated to be useful in inhibiting platelet aggregation and/or as cardiovascular agents for treatment of thrombotic conditions are disclosed in the following U.S. patents:
No. 4,593,029, issued June 3, 1986 to M C. Venuti et al, discloses .omega.-(N-imidazolyl) alkyl ethers of unsubstituted or substituted 1,2,3,5-tetrahydroimidazo [2,1--b]] quinazolines.
No. 4,588,741, issued May 13, 1986 to M. Nakane, discloses thiabicycloheptane substituted amino prostaglandin analogs.
No. 4,581,369, issued Apr. 8, 1986, to Mr. Tsuruda et al, discloses imidazole derivatives, such as .omega.-(2,4,6-trimethylphenyl)-3-(l 1-imidazolyl) benzenemethanol.
No. 4,575,512, issued Mar. 11, 1986 to S. E. Hall et al, discloses 7-oxabicycloheptane substituted oxa prostoglandin analogs.
No. 4,568,676, issued Feb. 4, 1986 to J. B. Smith discloses a method of inhibiting platelet aggregation by administering in combination a thromboxane systhetase inhibitor and an inhibitor of cyclic AMP phosphodiesterase, allegedly obtaining a synergistic effect.
No. 4,563,537, issued Jan. 7, 1986 to R. C. Larock, discloses thiophere-containing bicyclic prostaglandin endoperoxide analogs, synthesized by an addition reaction of a thienylpalladium to bicyclic alkenes.
Japanese patent application, public disclosure No. 227277/1984, published Dec. 20, 1984, discloses a method for producing a beverage containing guarana extract, wherein guarana seeds are placed in "potable alcohol" (presumably ethanol) for a number of days at room temperature, and the alcoholic extract is admixed with the main ingredients of the beverage, i.e. water and sweetening agents. The beverage may be carbonated, and contains about 1% alcohol.
Commonly used products for inhibiting blood platelet aggregation include aspirin, Dipyridamole, Anturan, and the like. However, many of the known products may exhibit other adverse physiological effects, and the antiaggregating action may be transient. Moreover, to the best of applicants' knowledge, there has been no suggestion in the prior art of an agent which has the ability to deaggregate platelet aggregates either in vitro or in vivo.
There is a definite need for a composition which can be produced simply and inexpensively and which can be administered orally or intravenously for deaggregation of platelet aggregates.